Topic 7 - modern genetics

Question 1

what doe PCR stand for?

Answer 1

polymerase chain reaction

Question 2

what is a PCR used for?

Answer 2

To amplify DNA (make lots of copies of the sample)

Question 3

What are the steps of a PCR?

Answer 3

denaturation:
heat DNA to 95 degrees
this splits strands by breaking h bonds
use enzyme from taq (can withstand heat)

annealing:
cool DNA to 55 degrees, primers bind to strands

extension:
heat to 72 degrees
add one polymerase strand to synthesise DNA from where the primer attached

Question 4

what machine does a PCR?

Answer 4

thermocycler

Question 5

what is a genome?

Answer 5

total genetic makeup of an organism

Question 6

what are introns/exons

Answer 6

introns- nucleotide sequences that DON’T code for a gene

exons- nucleotide sequences DO code for a gene

Question 7

why is a pH buffer used in PCR?

Answer 7

to keep the pH constant and maintain optimum pH level for enzyme activity

Question 8

what is an intron?

Answer 8

DNA that does not code for a protein

Question 9

within introns , there are satellites (STRs) - what are these?

Answer 9

satellites are found within introns , they are short sequences of DNA that are repeated many times.

Question 10

what are the two types of satellites (STRs) ?

Answer 10

micro satellites- 2-6 bases repeated 5-100 times

mini satellites 10-100 bases, repeated 50-several hundred times

Question 11

How do satellites (STRs) determine how closely related individuals are?

Answer 11

in a homologous pair of chromosomes, the same satellite is found at the same location - the thing that makes us unique is how often this is repeated.

The more closely related, the more similar the patternss in the size of satellites.

Question 12

What are the stages of DNA profiling?

Answer 12

PCR happens previously.

1. RESTRICTION ENDONUCLEASE ENZYMES
   used to cut DNA as they are made to have a specific active site which fits onto the specific intron where it wants to cut
2. EITHER SIDE OF SATELLITE IS CUT
3. GEL ELECTROPHORESIS
4. SOUTHERN BLOTTING
   alkaline buffer added (denatures DNA so it becomes single stranded)
   Nylon filler placed on gel- this draws the DNA fragments to it leaving them as ‘blots’ attached.
5. ADD GENE PROBES
   Gene probes are short single strand of DNA that have sequences complementary t sequences being sought.
   Probes have fluorescent marker attached
   bind with complementary DNA strands in a process called - hybridisation
6. GRAPH THE DATE COLLECTED FROM THE PROBE

Question 13

What is the name of the process when cells become specialised?

Answer 13

differentiation

Question 14

How do cells become specialised?

Answer 14

the genes we don’t want are switched off.

This means transcription doesn’t happen so RNA is not made , so gene is not translated.

Question 15

what are house keeping proteins?

Answer 15

housekeeping proteins are proteins present in most cells , for example ATP synthase

you can compare between cells by comparing the RNAs (they all have same DNA)

Question 16

what is a regulatory sequence and where is it found?

Answer 16

a regulatory sequence determines whether a gene is expressed or not -> these are found in our ‘junk DNA’ which consists of non-coding introns

regulatory sequences are either promoter or enhancer regions

Question 17

a regulatory sequence can be either a promotor or enhancer region

what does a promotor region do?

Answer 17

initiate transcription by enabling RNA polymerase to bind to the region they regulate

Question 18

a regulatory sequence can be either a promotor or enhancer region

what does an enhancer region do?

Answer 18

makes the gene more/less accessible by opening the chromatin structure (by loosening or tightening DNA around histome (when tightened, no gene)

Question 19

what is a transcription factor?

Answer 19

a protein that binds (in a complementary way) to a promotor region.

in either promotes/inhibits the binding site of RNA polymerase

Question 20

explain how splicing happens

Answer 20

happens during transcription.

full section of DNA copied onto mRNA (now known as pre-mRNA)

splice the pre-mRNA to remove introns (and some exons) - leaving only useful sections of exons

this is done by an enzyme called a spliceosome (this cuts, sticks and glues DNA)

now mature/functional mRNA can leave nucleus

Question 21

What are isoforms?

Answer 21

isoforms are different arrangements of exons- therefore one gene can produce several polypeptides because different exons can be removed during splicing.

Question 22

define epigenetics

Answer 22

the study of how your genome is expressed

Question 23

explain DNA methylation

Answer 23

happens to adenine or cytosine (cytosine in vertebrates)

done by enzyme methyltransferase

• CH3 (methyl) group added to the pyrimidine (in nitrogenous base)
• this can only happen at a CpG region (when a C and G are next to each other)

-occurs at promotor regions and prevents activation of RNA polymerase , so the gene is not transcribed

Question 24

how can methylation change your gene over time?

Answer 24

although your genome (DNA) does not change , your epigenome does vary.

more or less (demethylation) methyl groups are added during different stages of life.

they are hereditary

your epigenome can also be affected by factors of the environment , such as diet and smoking

Question 25

explain histone modification

Answer 25

histone and DNA is chromatin.

the histone can either be:

acetylated:
a CH3 joins to lycine or leucine
acetyl group transferred to molecules (donated by acetyl coenzyme A)
activates chromatin, allowing genes to be transcribed

methylated:
a CH3 joins to lycine or leucine
this tightens DNA, having an inhibitory effect

Question 26

stem cells

Answer 26

are capable of self renewal and of producing new cells that differentiate into other specialised types of cell

Question 27

define totipotent , pluripotent and multipotent cells

Answer 27

toti - can differentiate into any cell type, including umbilical cord and placenta

pluri- can differentiate into any cell , apart from umbilical cord and placenta

multi- can only differentiate into cells of a certain tissue

Question 28

pluripotent cells are affected by _________________ within blastocyst ?

Answer 28

location

Question 29

what are induced pluripotent stem cells? (iPS cells)

Answer 29

cell from body taken
add certain genes to the cell (genetic reprogramming)
induced pluripotent stem cell behaves like an embryonic stem cell

culture iPS in lab (grow by mitosis)
differentiation occurs - making all possible types of specialised cells.

Question 30

how is therapeutic cloning carried out?

Answer 30

remove nucleus from both body cell and ovum
combine body cell nucleus into ovum
multiply to make more pluripotent cells
separate and allow them to differentiate
transport into patient with no rejection issues.

Question 31

what are the two methods of making recombinant DNA?

Answer 31

method 1:
mRNA purified from cell of inetrest
use reverse transcriptase to turn mRNA into cDNA (complimentary DNA)
DNA polymerase used to make the other strand of DNA

method 2:
isolate gene by restriction endonucleases
this cuts a specific base sequence of DNA
creates ‘sticky ends’ that can clip back together

NEXT (after either method)
plasmids are vector for gene to be inserted into another organism
plasmid’s sticky ends are complimentary to sticky ends of DNA
plasmid is cut with restriction endonuclease enzyme and DNA ligase sticks DNA into plasmid by joining up sugar phosphate backbone.
Reincorporate plasmid into host cell nucleus.